Supplementary Material to Rouvinen-Watt Et Al., Page 1

Supplementary Material to Rouvinen-Watt et al., page 1

Supplementary Material to

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis by Rouvinen-Watt et al.

CONTENT

Doc S1 of Supplementary Material…………………………………………….……………….1

Author contributions 1

RNA extraction 1

Reverse transcription 2

qPCR protocol 2

Validation 2

Data analysis 2

MIQE checklist 3

Table S1 of Supplementary Material…………………………………………….……………..7

Table S2 of Supplementary Material…………………………………………….……………..7

Table S3 of Supplementary Material…………………………………………….……………..8

References to Supplementary Material…………………………………………….…………..8

Author contributions

K. Rouvinen-Watt, P. Nieminen, and A.-M. Mustonen planned this research and KRW oversaw all experimental procedures. C. Pal, T. Martin, L. Harris, D. Kryzskaya, and V. Kärjä carried out the laboratory analyses. Data analysis was performed by KRW, T. Astatkie, CP, TM, and PN. R. Tammi and M. Tammi contributed to the interpretations of the data. Manuscript writing was led by KRW, with contributions from all co-authors.

RNA extraction

Mink livers were obtained (by macrodissection) within 15–25 min from time of death. Samples were immediately snap frozen in liquid nitrogen and kept at –85°C until RNA extraction, six months later. Total RNA content was extracted from approximately 30 mg tissue, with homogenization using the Kimble® Kontes Pellet Pestle and QIAShredder homogenizers. RNA was treated with RNase free DNase I (Qiagen, Toronto, ON, Canada) using the manufacturer’s on-column treatment protocol. There was an average yield of 2.28 ± 0.06 µg RNA/mg tissue. Sample purity was assessed by an A260:A280 ratio of 1.8–2.1.

Reverse transcription

Total RNA (1.0 µg) was reverse transcribed with 2.5 U/µL MultiScribe™ Reverse Transcriptase in a reaction volume of 20 µL at 37°C for 120 min, using the High Capacity cDNA Reverse Transcription Kit with RNase inhibitor, catalog# 4374966 (Applied Biosystems, Carlsbad, CA, USA). 0.1X and 0.01X dilutions of cDNA were prepared in 10 mM Tris-Cl, pH=7.6, and stored at –30°C. GAPDH qPCR run on a set of no reverse transcription controls showed no genomic contamination in 42 samples, minor contamination (Cq=31.2) in one sample, and trace contamination in the remaining 17 samples (Cq>35).

qPCR protocol

Primers, purified by reverse-phase cartridge separation, were purchased from Sigma (Sigma-Aldrich Canada Co, Oakville, ON, Canada). Assays were run in a reaction volume of 10 µL, using 1 µL of 0.1X diluted cDNA, using 384-well white plates, catalog #04729749001 (Roche Diagnotics Canada, Laval, QC, Canada). Each sample was run with three technical replicates at the qPCR stage, and each treatment group included five technical replicates. All assays except GRP78 were run with 1X GoTaq® Flexi buffer, 2.5 mM MgCl2, 0.2 mM dNTP (each), 0.8 U GoTaq® HotStart Polymerase (Promega, Fitchburg, WI, USA), and 1X EvaGreen® (Biotium Inc, Hayward, CA, USA). The GRP78 assay was run using 1X SsoFastTM EvaGreen® mastermix (Bio-Rad Laboratories Inc., Hercules, CA, USA), 500 nM primers, with a hot start at 95°C for 2 min followed by 35 cycles of denaturation at 95°C for 5 s, combined annealing and extension at 58°C for 20 s.

Validation

The specificity of each qPCR assay was confirmed by verification of the size of the amplicon on agarose gel electrophoresis (gel photographs available on request), and the presence of a single peak in a melt curve. No amplification was seen in the no template controls (n=6). The limit of detection for each assay was at or below the lower limit of the linear dynamic range as reported in Table S3, as shown by a linear standard curve to this point. To test for inhibition, qPCR results of standards spiked with pooled cDNA (0.1X dilutions) were compared with those of pooled cDNA (0.1X dilution) alone, and standards run separately. No inhibition was seen.

Data analysis

Lightcycler® 480 Software, release 1.5.0.39 (Roche, Indianapolis, IN, USA) was used to obtain quantification cycles (Cq) results for each sample, employing the “second derivative maximum” method. Each qPCR run included a standard curve (a 5-fold dilution series from a known amount of target cDNA, created from synthetic copy RNA), which was used to calculate the expression of the target gene in each sample. Each run was examined for anomalous amplification curves, and all were within acceptable norms, so that no outliers were removed. To normalize each sample, the geometric mean of the replicate concentrations of a target gene was divided by the geometric mean of the replicate concentrations of the reference gene.

Supplementary Material to Rouvinen-Watt et al., page 1

MIQE Checklist according to Bustin et al. (2009)
ITEM TO CHECK / IMPORTANCE / CHECKLIST / WHERE IN THE MANUSCRIPT
EXPERIMENTAL DESIGN
Definition of experimental and control groups / E / Yes / Methods and materials: Animal experiment
Number within each group / E / Yes / Methods and materials: Animal experiment
Assay carried out by core lab or investigator's lab? / D / Yes / All experiments carried out in investigator’s lab.
Acknowledgement of authors' contributions / D / Yes / S1: Author contributions
SAMPLE
Description / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Volume/mass of sample processed / D / Yes / S1: RNA extraction
Microdissection or macrodissection / E / Yes / S1: RNA extraction
Processing procedure / E / Yes / S1: RNA extraction
If frozen - how and how quickly? / E / Yes / S1: RNA extraction
If fixed - with what, how quickly? / E / Yes / S1: RNA extraction
Sample storage conditions and duration (especially for FFPE samples) / E / Yes / S1: RNA extraction
NUCLEIC ACID EXTRACTION
Procedure and/or instrumentation / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Name of kit and details of any modifications / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Source of additional reagents used / D / n/a
Details of DNase or RNase treatment / E / Yes / S1: Validation
Contamination assessment (DNA or RNA) / E / Yes / S1: Reverse transcription
Nucleic acid quantification / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Instrument and method / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Purity (A260/A280) / D / Yes / S1: RNA extraction
Yield / D / Yes / S1: RNA extraction
RNA integrity method/instrument / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
RIN/RQI or Cq of 3' and 5' transcripts / E / ND
Electrophoresis traces / D / Yes / Available on request
Inhibition testing (Cq dilutions, spike or other) / E / Yes / S1: RNA extraction
REVERSE TRANSCRIPTION
Complete reaction conditions / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction and S1
Amount of RNA and reaction volume / E / Yes / S1: Reverse transcription
Priming oligonucleotide (if using GSP) and concentration / E / N/A
Reverse transcriptase and concentration / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction and S1: Reverse transcription
Temperature and time / E / Yes / S1: Reverse transcription
Manufacturer of reagents and catalogue numbers / D / Yes / S1: Reverse transcription
Cqs with and without RT / D* / Yes / S1: Reverse transcription
Storage conditions of cDNA / D / Yes / S1: Reverse transcription
qPCR TARGET INFORMATION
If multiplex, efficiency and LOD of each assay. / E / N/A
Sequence accession number / E / Yes / Table 1
Location of amplicon / D / Yes / S1: Table S1
Amplicon length / E / Yes / Table 1
In silico specificity screen (BLAST, etc) / E / Yes / Primer BLAST run for all targets (Ye et al. 2012)
Pseudogenes, retropseudogenes or other homologs? / D / No / Information unavailable for mink.
Sequence alignment / D / No
Secondary structure analysis of amplicon / D / Yes / All amplicons analyzed using Mfold (Zuker 2003)
Location of each primer by exon or intron (if applicable) / E / Yes / S1: Table S1
What splice variants are targeted? / E / No / Splice variation information unavailable for mink.
qPCR OLIGONUCLEOTIDES
Primer sequences / E / Yes / Table 1
RTPrimerDB Identification Number / D / N/A
Probe sequences / D** / N/A
Location and identity of any modifications / E / Yes / None
Manufacturer of oligonucleotides / D / Yes / S1: qPCR protocol
Purification method / D / Yes / S1: qPCR protocol
qPCR PROTOCOL
Complete reaction conditions / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Reaction volume and amount of cDNA/DNA / E / Yes / S1: qPCR Protocol
Primer, (probe), Mg++ and dNTP concentrations / E / Yes / S1: Table S2 and qPCR protocol
Polymerase identity and concentration / E / Yes / S1: qPCR protocol
Buffer/kit identity and manufacturer / E / Yes / S1: qPCR protocol
Exact chemical constitution of the buffer / D / No / Not available from manufacturer.
Additives (SYBR Green I, DMSO, etc.) / E / Yes / S1: qPCR protocol
Manufacturer of plates/tubes and catalog number / D / Yes / Methods and materials: Quantitative real-time polymerase chain reaction and S1: qPCR protocol
Complete thermocycling parameters / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction, S1: Table S2, and S1: qPCR protocol
Reaction setup (manual/robotic) / D / Yes / Methods and materials: Quantitative Real-Time Polymerase Chain Reaction
Manufacturer of qPCR instrument / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
qPCR VALIDATION
Evidence of optimization (from gradients) / D / Yes / Available on request
Specificity (gel, sequence, melt, or digest) / E / Yes / S1: Validation
For SYBR Green I, Cq of the NTC / E / Yes / S1: Validation
Standard curves with slope and y-intercept / E / Yes / S1: Table S3
PCR efficiency calculated from slope / E / Yes / S1: Table S3
Confidence interval for PCR efficiency or standard error / D / No
r2 of standard curve / E / Yes / S1: Table S3
Linear dynamic range / E / Yes / S1: Table S3
Cq variation at lower limit / E / Yes / S1: Table S3
Confidence intervals throughout range / D / No
Evidence for limit of detection / E / Yes / S1: Validation
If multiplex, efficiency and LOD of each assay. / E / N/A
DATA ANALYSIS
qPCR analysis program (source, version) / E / Yes / S1: Data analysis
Cq method determination / E / Yes / S1: Data analysis
Outlier identification and disposition / E / Yes / S1: Data analysis
Results of NTCs / E / Yes / S1: Validation
Justification of number and choice of reference genes / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction
Description of normalisation method / E / Yes / Methods and materials: Quantitative real-time polymerase chain reaction and S1: Data analysis
Number and concordance of biological replicates / D / Yes / Methods and materials: Animal experiment and S1: qPCR protocol
Number and stage (RT or qPCR) of technical replicates / E / Yes / S1: qPCR protocol
Repeatability (intra-assay variation) / E / Yes / S1: Table S3
Reproducibility (inter-assay variation, %CV) / D / No
Power analysis / D / No
Statistical methods for result significance / E / Yes / Methods and materials: Statistical analysis
Software (source, version) / E / Yes / Methods and materials: Statistical analysis
Cq or raw data submission using RDML / D / No

Table S1 Target information for qPCR primer pairs.

Target gene / Sequence accession number / Location of amplicon / Amplicon length / Location of forward primer (human exon) / Location of reverse primer (human exon)
GAPDH / AF076283 / 36-116 / 84 / 7 / 7
GRP78 / HQ003898 / 501-611 / 111 / 8 / 8
LPL / AJ223493 / 1813-1908 / 96 / 10 / 10
MCP-1 / HQ163895 / 149-266 / 118 / 2 / 3
TNF-a / GU327784 / 81-205 / 125 / 3 / 4

Table S2 Thermocycling parameters for qPCR assays.

Target gene / Concentration primers (nM) / Number of cycles / Annealing temperature (°C) / Denaturation time (s) / Annealing time (s) / Extension time (s)
GAPDH / 400 / 40 / 60 / 10 / 20 / 30
LPL / 400 / 45 / 60 / 10 / 20 / 30
MCP-1 / 400 / 45 / 60 / 30 / 30 / 30
TNF-a / 400 / 45 / 62 / 30 / 30 / 30

Table S3 qPCR validation for qPCR assays.

Target / Standard curve / Linear dynamic / Cq variation at / Repeatability (intra-assay
gene / Slope / Y-intercept / Efficiency (%) / r2 / range (copies) / lower limit / variation) (CV of Cq, %)
GAPDH / –3.481 / 41.69 / 93.8 / 0.9999 / 103-108 / 0.07 / 0.10
GRP78 / –3.542 / 42.28 / 91.6 / 0.9990 / 103-108 / 0.00 / 0.25
LPL / –3.513 / 43.29 / 92.6 / 0.9988 / 102-108 / 0.82 / 0.19
MCP-1 / –3.465 / 47.41 / 94.4 / 0.9968 / 103-108 / 0.43 / 0.46
TNF-a / –3.455 / 40.05 / 94.7 / 0.9968 / 25-108 / 0.06 / 1.11

References to Supplementary Material

Bustin SA, Benes V, Garson JA et al (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin Chem 55:611–622

Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134

Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415